Method for treating inflammatory disease in humans

ABSTRACT

Benzothiophene, benzofuran and indole-thiazepinones, oxazepinones and diazepinones as well as methods of preparation thereof are described as agents which inhibit leukocyte adherence to vascular endothelium and, as such, are effective therapeutic agents for treating inflammatory diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. Ser. No. 08/207,330 filed Mar. 7,1994, now abandoned.

BACKGROUND OF THE INVENTION

The present invention is for novel benzothiophene, benzofuran and indolethiazepinones, oxazepinones and diazepinones and pharmaceuticallyacceptable salts thereof, used to prevent the adhesion of leukocytes toendothelial cells. Leukocyte adherence to vascular endothelium isintegral to the pathogenesis of inflammation. The adhesion processprecedes transendothelial migration of leukocytes into surroundingtissue and ensuing tissue damage. Compounds that can block this initialadhesive interaction are expected to have efficacy in the treatment ofinflammatory diseases such as rheumatoid arthritis, osteoarthritis,asthma, and psoriasis. Other indications would include but are notlimited to adult respiratory distress syndrome, reperfusion injury,ischemia, ulcerative coliris, vasculitides, atherosclerosis,inflammatory bowel disease and tumor metastases.

Adhesion receptors are organized into three main families: theselectins, the immunoglobulin superfamily, and the integrins (Nature,346:426 (1990)). Members of all three classes are involved in mediatingleukocyte adhesion during inflammation (for reviews of this area see:Thrombosis and Hemostasis, 65(3);223 (1991), Clinical and ExperimentalAllergy, 20:619 (1990), Transplantation, 48:727 (1989), BiochemicalPharm, 40 (8):1683 (1990)). Endothelial leukocyte adhesion molecule-1(ELAM-1 or E-selectin) is a member of the selectin family ofglycoproteins that promote cell-cell adhesion. E-selectin is reported tobe maximally expressed on the surface of endothelial cells 4 hours afterstimulation of the endothelial cells with cytokines, such asinterleukin-1 (IL-1) or tumor necrosis factor α (TNF-α) or otherinflammatory mediators, such as lipopolysaccharide (LPS) (Pro. Nat.Acad. Sci., 84:9238 (1987).

Intercellular adhesion molecule-1 (ICAM-1) is a member of theimmunoglobulin superfamily. It is also upregulated with maximumexpression occurring 12-24 hours after stimulus. It has been shown that4 hours after the endothelial cells are stimulated with an inflammatorymediator both E-selectin and ICAM-1 are present on the cell surface (J.Clin. Invest., 82: 1746 (1988) and J. Immun., 137:1893 (1986), Blood,78:2721 (1991)).

The benzothiophene, benzofuran and indole thiazepinones, oxazepinonesand diazepinones of the present invention have been shown to inhibit theadhesion of neutrophils to human umbilical vein endothelial cells(HUVECS) stimulated with TNFα in an in vitro assay.

SUMMARY OF THE INVENTION

Accordingly, the present invention is a compound of the Formula (I) or apharmaceutically acceptable acid addition salt thereof: ##STR1## whereinR₁, R₂, R₃, and R₄ are each independently hydrogen, hydroxy, halogen,lower alkyl, lower alkoxy, benzyloxy, trifluoromethyl, nitro, or --NR₈R₉, in which

R₈ and R₉ are each independently hydrogen or lower alkyl;

R₅ and R₆ are each independently hydrogen, lower alkyl or phenyl;

X is O, (O)_(n) or NR₇ ;

Y is O, S(O)_(n) or NR₈ ;

R₇ is hydrogen, lower alkyl, phenyl, benzyl, CH₂ OR₈ or lower alkyl,phenyl, benzyl substituted with halo;

R₈ is hydrogen, lower alkyl or phenyl;

n is an integer of 0, 1 or 2;

with the provisos that

1) when X is NH, Y is NH, R₁ is H, R₃ is H and R₄ is Br, R₂ is notmethyl;

2) when X is NH, Y is NH, R₁, R₃ and R₄ are H, R₂ is not methoxy orethoxy, and

3) when X is NH, Y is S, at least one of R₁, R₂, R₃ and R₄ is not H.

The present invention includes pharmaceutical compositions comprising atherapeutically effective amount of a compound of the Formula I above,together with a pharmaceutically acceptable carrier.

A third aspect of the present invention is a method of treating diseasesmediated by inhibiting the adhesion of leukocytes to endothelial cellscomprising administering to a host in need thereof a pharmaceuticalcomposition containing a compound of Formula I above in unit dosageform.

A preferred embodiment is a method for treating inflammatory disease inhumans comprising administering an antiinflammatory amount of a compoundof Formula I.

DETAILED DESCRIPTION

The terms used in defining the compounds of Formula I of the presentinvention are defined as follows:

Lower alkyl and lower alkoxy mean a straight or branched alkyl or alkoxygroup having 1 to 4 carbon atoms and includes, for example, methyl,ethyl, propyl, i-propyl, or otherwise referred to as (methyl)ethyl, andt-butyl or otherwise referred to as 1,1-(dimethyl)ethyl, andcorrespondingly, for example, methoxy, ethoxy, i-propoxy, or otherwisereferred to as 1-(methyl)ethoxy and the like.

Halogen includes fluorine, chlorine, bromine, or iodine.

The compounds of the Formula I are capable of further formingpharmaceutically acceptable acid addition salts. All of these forms arewithin the scope of the present invention.

Pharmaceutically acceptable acid addition salts of the compounds ofFormula I include salts derived from inorganic acids such ashydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydriodic,hydrofluoric, phosphorous, and the like, as well as the salts derivedfrom nontoxic organic acids, such as aliphatic mono- and dicarboxylicacids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids,alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonicacids, etc. Such salts thus include sulfate, pyrosulfate, bisulfate,sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, trifluoroacetate, propionate, caprylate, isobutyrate,oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate,mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate,lactate, maleate, tartrate, methanesulfonate, and the like. Alsocontemplated are salts of amino acids such as arginate and the like andgluconate, galacturonate, N-methyl glutamine (see, for example, Berge S.M., et al., "Pharmaceutical Salts," Journal of Pharmaceutical Science,66:1-19 (1977)).

The acid addition salts of said basic compounds are prepared bycontacting the free base form with a sufficient amount of the desiredacid to produce the salt in the conventional manner. The free base formmay be regenerated by contacting the salt form with a base and isolatingthe free base in the conventional manner. The free base forms differfrom their respective salt forms somewhat in certain physical propertiessuch as solubility in polar solvents, but otherwise the salts areequivalent to their respective free base for purposes of the presentinvention.

Certain of the compounds of the present invention can exist inunsolvated forms as well as solvated forms, including hydrated forms. Ingeneral, the solvated forms, including hydrated forms, are equivalent tounsolvated forms and are intended to be encompassed within the scope ofthe present invention.

A preferred embodiment of the present invention is a compound of FormulaI, wherein R₁, R₃ and R₄ are hydrogen and R₂ is as defined above.

A more preferred embodiment of the present invention is a compound ofFormula I, wherein R₁, R₃ and R₄ are hydrogen; R₂ is hydrogen or loweralkoxy; X is O, S(O)_(n) or NR₇ ; Y is O or S(O)_(n) ; R₇ is hydrogen orlower alkyl, and n is 0, 1, or 2.

Particularly valuable are:

2,3-dihydro-9-methoxy-[1]benzothieno[2,3-f]-1,4-thiazepin-5(4H)-one,2,3-dihydro-[1]benzothieno[2,3-f]-1,4-oxazepin-5(4H)-one,2,3-dihydro-9-methoxy-[1]benzothieno[2,3-f]-1,4-thiazepin-5(4H)-one-1-oxide, 3,4-dihydro-9-methoxy-6-methyl-2H-1,4-oxazepino[6,7-b]-indol-5(6H)-one,2,3-dihydro-1H-benzothieno-[3,2-e]-1,4-diazepine-5-one,2,3-dihydro-9-methoxy-1H-benzothieno-[2,3-f] -1,4-oxazepine-5-one,2,3-dihydro-9-methoxy-6-oxide-1H-benzothieno-[2,3-f]oxazepine-5-one,2,3-dihydro-9-methoxy-2-methyl-1H-benzothieno-[2,3-f]-1,4-oxazepine-5-one,2,3-dihydro-7,8,9,10-tetrachloro-1H-benzothieno[2,3-f]-1,4-oxazepine-5-one,3,4-dihydro-8-nitro-6-tert.-butyl-2H-1,4-oxazepine[6,7-b]indol-5(6H)-one,3,4-dihydro -9-isopropoxy-6-phenoxymethyl-2H-1,4-oxazepine[6,7-b]indol-5(6H)-one hydrochloride,3,4-dihydro-8,10-dibromo-6-(3-chlorobenzyl-2H-1,4-oxazepino[6,7-b]indol-5(6H)-one,2,3-dihydro-8-chloro-1H-benzofurano[2,3-f]-1,4-oxazepine-5-one,methanesulfonate,2,3-dihydro-1,2,3-trimethyl-1H-benzofurano[3,2-e]-1,4-diazepine-5-one,and 2,3-dihydro-3-hexyl-1H-benzofurano[2,3-f]-1,4-thiazepine-5-one.

For medical use, the amount required of a compound of Formula I or apharmacologically acceptable acid addition salt thereof to achieve atherapeutic effect will, of course, vary both with the particularcompound, the route of administration, the mammal under treatment, andthe particular disorder of disease concerned. In a preferred embodiment,the invention provides a method for treating humans suffering frominflammatory disease, such as arthritis or swelling comprisingadministering an antiinflammatory effective amount to the subject inneed of treatment. A suitable dose of a compound of Formula I or apharmacologically acceptable acid addition salt thereof for a mammalsuffering from, or likely to suffer from any condition as describedhereinbefore is 0.1 μg to 500 mg of the compound per kilogram bodyweight. In the case of systemic administration, the dose may be in therange of 0.5 to 500 mg of the compound per kilogram body weight, themost preferred dosage being 0.5 to 50 mg/kg of mammal body weightadministered two to three times daily. In the case of topicaladministration, e.g., to the skin or eye, a suitable dose may be in therange 0.1 ng to 100 μg of the compound per kilogram, typically about 0.1μg/kg.

In the case of oral dosing for the treatment or prophylaxis of arthritisor inflammation in general, due to any cause, a suitable dose of acompound of Formula I or a physiologically acceptable acid addition saltthereof, may be as specified in the preceding paragraph, but mostpreferably is from 1 mg to 10 mg of the compound per kilogram, the mostpreferred dosage being from 1 mg to 5 mg/kg of mammal body weight, forexample, from 1 to 2 mg/kg.

It is understood that the ordinarily skilled physician or veterinarianwill readily determine and prescribe the effective amount of thecompound to prevent or arrest the progress of the condition for whichtreatment is administered. In so proceeding, the physician orveterinarian could employ relatively low doses at first, subsequentlyincreasing the dose until a maximum response is obtained.

While it is possible for an active ingredient to be administered alone,it is preferable to present it as a pharmaceutical formulationcomprising a compound of Formula I or a pharmacologically acceptableacid addition salt thereof and a pharmacologically acceptable carriertherefor. Such formulations constitute a further feature of the presentinvention.

The formulations, both for veterinary and for human medical use, of thepresent invention comprise an active ingredient in association with apharmaceutically acceptable carrier therefor and optionally othertherapeutic ingredient(s). The carrier(s) must be `acceptable` in thesense of being compatible with the other ingredients of the formulationsand not deleterious to the recipient thereof.

The formulations include those in a form suitable for oral, pulmonary,ophthalmic, rectal, parenteral (including subcutaneous, intramuscular,and intravenous), intraarticular, topical, nasal, or buccaladministration. Such formulations are understood to include long-actingformulations known in the art.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods may include the step of bringing the active ingredient intoassociation with the carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active ingredient into association with a liquidcarrier or a finely divided solid carrier or both, and then, ifnecessary, shaping the product into the desired formulation.

Formulations of the present invention suitable for oral administrationmay be in the form of discrete units such as capsules, cachets, tablets,or lozenges, each containing a predetermined amount of the activeingredient; in the form of a powder or granules; in the form of asolution or a suspension in an aqueous liquid or nonaqueous liquid; orin the form of an oil-in-water emulsion or a water-in-oil emulsion. Theactive ingredient may also be in the form of a bolus, electuary, orpaste.

The usefulness of the compounds of the present invention as inhibitorsof leukocyte adherence to vascular endothelium and thus in treatinginflammatory-related diseases or conditions may be demonstrated by theireffectiveness in various standard test procedures. A description of eachprocedure and exemplary test results follows.

Protocol for the Intercellular Adhesion Molecule-1/HUVEC ExpressionAssay (ICAM-1) and the E-Selectin/HUVEC Expression Assay (ESEL)

Cell culture

Human umbilical cord endothelial cells (HUVECs) from Clonetics werepurchased in T-25 tissue culture flasks and allowed to grow for 1-3 daysafter arrival at 37° C. and 5% carbon dioxide. The HUVECs were thensplit by rinsing the T-25 with 10 mL of a 0.025% trypsin/0.01% EDTA for5-10 seconds, pouring off the rinse solution. Another 10 mL of thetrypsin/EDTA solution was added and the cells were agitated for 2-4minutes while rapping on the side of the flask with a pencil eraser. Thecontents of the flask were then poured into a 50 mL centrifuge tubecontaining 40 mL of media. The media was endothelial basal mediapurchased from Clonetics containing hydrocortisone (2 mg/L), epidermalgrowth factor (0.05 ug/L), bovine brain extract (12 mg/L) and heatinactivated fetal calf serum (6%) from Hyclone. The cells werecentrifuged at 15° C. for 10-15 minutes, the supernatant drained off,and the cells resuspended with fresh media. Cells were washed in anidentical manner a second time and then seeded into 96 well tissueculture plates.

Cytokine Stimulation

Within 5 days after reaching confluence the cells were stimulated withtumor necrosis factor alpha (TNFα) (Genzyme) to obtain a final mediaconcentration of 140 U/mL and allowed to incubate at 37° C. for 4 hours.After the 4 hour incubation, the media was removed and stored foranalysis of chemokine production. The cells were washed 3 times withcalcium and magnesium free phosphate buffered saline. The monocultureswere then fixed by adding 10% buffered formalin to the wells for 15minutes. After fixing, the cells were washed 3 times with Dulbecco'sModified Eagle Media (Gibco) containing 2% bovine serum albumin(DMEM/2%BSA) and refrigerated overnight.

The ELISA

Murine monoclonal anti-human ICAM-1 (R & D Systems, Cat. No. BBA-4) ormurine monoclonal anti-human E-selectin (R & D, Cat. No. BBA-2)dissolved in DMEM/2% BSA were added to each well at 0.5 μg/mL andallowed to incubate at 37° C. for 2 hours. HUVEC monocultures were thenwashed 4 times with DMEM/2%BSA. A peroxidase conjugated sheep anti-mouseIgG (Cappel) was added (1:3,000 dilution) and allowed to incubate 1 hourat 37° C. The cells were then washed 4 times with DMEM. A color reagent(Biorad) was added to the fixed cells and incubated 15 minutes at roomtemperature. The reaction was stopped with a 2% oxalic acid solution andthe absorbance read at 414 nm on a titertek plate reader.

Compound Testing

Compounds were dissolved in DMSA at a concentration of 30 mmol anddiluted with media to obtain final testing concentrations. HUVECsreceived compound dissolved in media 30 minutes before the TNFαchallenge. The absorbance of non-stimulated HUVECs was subtracted fromthe absorbance values of TNFα stimulated cells before percent inhibitionwas determined. Percent inhibition was determined by comparing theabsorbance of vehicle treated cells with drug treated cells. IC₅₀ s weredetermined using linear regressoin analysis.

METHOD FOR DETERMINING THE INHIBITION OF HUMAN NEUTROPHIL ADHESION TOTHE TNF-α STIMULATED HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (ECA)

Cell Culture

Second passage HUVEC (Clonetics Corporation, San Diego, Calif., CC-2617)were seeded into Corning (Corning glass works, Corning, N.Y.) 96-wellcell culture plates at approximately 5×10³ cells/well and grown toconfluency in supplemented endothelial basal medium (EBM MCDB-131,Clonetics, 10 ng/mL EGF, 1 μg/mL hydrocortisone, 0.4% bovine brainextract, 5% Fetal Bovine Serum). One day prior to running the assay,typically 3 days postseeding, the cultures were refed with 0.2 mL/wellsupplemented EBM (S-EBM).

Preparation of Test Compounds

Test compounds were prepared as 10 mL stock solutions at a concentrationof 1.0 mM. The compounds were initially solubilized in 0.1 mL DMSOfollowed by the addition of 9.9 mL S-EBM. The drug preparations werethen diluted in one step to a concentration of 66.6 μM. Solubilizationsand dilutions were performed in polystyrene containers.

Stimulation of HUVEC

Recombinant human tumor necrosis factor-α (TNF, Genzyme, Boston, Mass.,code TNF-H) was prepared at 400 U/mL in S-EBM. Stock TNF was prepared to20,000 U/mL in Delbecco's phosphate buffered saline (PBS, Gibco, GrandIsland, N.Y.) plus 0.1% BSA and stored at -70° C. HUVEC were washed onetime with 0.2 mL warm unsupplemented EBM and then stimulated for 4 hoursat 37° C. with 200 U/mL TNF in the presence of 33.3 μM test compound.This was accomplished by adding 0.1 mL of 400 U/mL TNF and 0.1 mL 66.6μM test compound. These additions were done slowly as to not disrupt theHUVEC monolayer. Each compound was tested in six wells. Unstimulated(vehicle control) and TNF-stimulated without test compound treatmentswere also run in each plate.

Labeling of Neutrophils

One hour prior to adding the neutrophils to the HUVEC, neutrophils(5×10⁶ /mL) were labeled for 30 minutes at 37° C. with 5 μM calcein-AM(Molecular Probes, Eugene, Oreg.) in Hanks' balanced salt solution plus0.45% BSA. Stock calcein was prepared to 5 mM in anhydrous DMSO andstored desiccated at -20° C. At the end of the incubation the cells werewashed two times in cold HBSS and resuspended to a final concentrationof 1×10⁶ cells/mL in supplemented EBM.

Addition of Neutrophils to HUVEC

At the end of the 4-hour stimulation and immediately prior to theaddition of the neutrophils to the HUVEC monolayer, the plates werewashed with 0.2 mL warm unsupplemented EBM to remove TNF and drug.Neutrophils (1×10⁵ cells) were slowly added to each of the treated wellsand incubated for 30 minutes at 37° C. At the end of the incubation theplates were washed two times with 0.2 mL warm unsupplemented EBMfollowed by a final addition of 0.1 mL for plate scanning.

Determination of Relative Fluroescence

The relative fluroescence was determined using a Millipore Cytofluor 300system (excitation=480, emission=530, sensitivity=4).

Calculations

The assay was considered valid if the TNF-stimulation of the HUVECresulted in a 300% increase in neutrophil adherence over adherence tounstimulated HUVEC. Results were expressed as means of percentinhibitiion of TNF-stimulated adherence. ##EQU1##

Some of these compounds were tested at concentrations of 33.3 μM, 10.0μM, 3.3 μM, and 1.0 μM to determined IC₅₀ values. Linear regressionanalysis of the means of the inhibition values were used to determinethe IC₅₀.

The results obtained with certain compounds of the present invention areshown in Table I.

                  TABLE I                                                         ______________________________________                                         ##STR2##                                                                     2,3-Dihydro-9-methoxy-[1]benzothieno[2,3-f]-                                  1,4-thiazepin-5(4H)-one                                                                                          ICAM/                                                                         ESEL                                                                          (IC.sub.50                                 Ex-                                or %    OM-10                              am-                          ECA   Inhib @ (IC.sub.50                         ple  R.sub.2                                                                              X       Y        (IC.sub.50)                                                                         30 μM)                                                                             μM)                             ______________________________________                                        1    OMe    S       S        5.2   3.1/1.3 .21                                2    H      S       O              42%/40% >30                                4    OMe    NMe     O              14.7/14.2                                  5    H      S       NH             64%/47%                                    6    OMe    S       O              3.1/7.5                                    7    OMe    SO      O              30%/30%                                    8    OMe    S       O (2-methyl)   3.8/5.3                                    ______________________________________                                    

The invention compounds have also demonstrated activity in standard invivo assays utilized to measure their ability to inhibit neutrophilinflux, and accordingly their utility to treat conditions ofinflammation. In one test called the Reverse Passive Arthus PleurisyAssay, male outbred Wistar rats (220-245 g, Charles River Laboratories)were fasted for 16 to 18 hours. Vehicle (1:1, ethanol:saline) or aninvention compound dissolved in vehicle was administered IV. The animalswere lightly anesthetized with ether and given an IV injection of 2.5 mgbovine serum albumin (BSA) in saline. Immediately following the IVinjection, a small incision was made between the ribs, and 0.2 mL of arabbit IgG fraction anti-BSA (10 mg/mL in Dulbecco's Phosphate BufferedSaline (PBS)) in PBS was injected into the pleural cavity using a20-gauge oral dosing needle. The incision was then closed with a 9-mmstainless steel wound clip. Four hours later the animals were euthanizedwith carbon dioxide and the pleural cavity flushed with 2 mL of a 0.325%phenol red solution in PBS. The exudate-buffer were removed from thepleural cavity for analysis. White blood cells (>90% neutrophils) werecounted using a Coulter counter. The pleural exudate volume was measuredby a dye dilution method (Carter G. W., et al., J. Pharm. Pharmacol.34:66-67 (1982)). Drug treatment groups were compared to avehicle-treated group and statistical significance determined usingStudent's t-test.

When the compound of Example 1 was evaluated in the above test, itexhibited the following inhibitions:

    ______________________________________                                                  Percent Inhibition                                                                         Percent Inhibition                                     Dose (mg/kg)                                                                            of Exudate   of Neutrophil Influx                                   ______________________________________                                        0.3       40.5         18.6                                                   1.0       28.4          8.9                                                   3.0       28.2         15.9                                                   ______________________________________                                    

In another in vivo assay called the Thioglycollate-induced neutrophilinflux assay, female Balb/c in-bred mice are housed in groups of sevenwith free access to food and water throughout the study. The animals areorally dosed with vehicle (0.5% hydroxypropyl methyl cellulose with 0.2%Tween 80) or invention compound dissolved or suspended in vehicle. Onehour after oral administration, the mice are anesthetized by diethylether inhalation and intraperitoneally injected with 1.0 mL of 3%thioglycollate medium in saline. Two hours post-thioglycollateinjection, the animals are euthanized by carbon dioxide asphyxiation andinjected with 6 mL Dulbecco's PBS containing 10 U/mL sodium heparin and0.1% BSA. The peritoneal cavity is massaged and an incision is made intothe cavity to allow the fluid to be collected into 15-mL centrifugetubes. An aliquot is removed from each animal and the total number ofcells in each aliquot are counted using a Coulter Counter (Model ZBi,Coulter Instruments, Hialeah, Fla.). A second aliquot is removed formicroscopy using the Cytospin 2 (Shandon Inc., Pittsburgh, Pa.), andsubsequent staining (modified Wright's stain) is performed. Hematologicdifferentials are performed to determine the percentage of neutrophilswhich have extravasated to the peritoneal cavity.

When the compound of Example 1 was evaluated in this assay, it exhibited26.1% inhibition of neutrophil influx at 10 mg/kg, 31.9% inhibition at30 mg/kg, and 4.3% inhibition at 100 mg/kg.

The compounds of the present invention may be prepared by the followingmethods.

The first general approach requires as starting materials the 3-hydroxy,thiol, or aminobenzo[b]thiophene, benzofuran or indole -2-carboxylateesters of structure 1 (Scheme 1). The3-hydroxybenzo[b]thiophene-2-esters are prepared as documented [ConnorD. T., et al., J. Med. Chem., 35:958 (1992)]. The3-thio-benzo[b]thiophene-2-carboxylate esters are prepared by treatmentof the analogous 3-chloro derivative [Connor D. T., et al., J. Med.Chem., 35:958 (1992)] with thioacetamide in the presence of a base suchas 1,8-diaza-bicyclo[5.4.0]-undec-7-ene (DBU) and a solvent such asN,N'-dimethylformamide or tetrahydrofuran. The3-amino-benzo[b]thiophene-2-carboxylate esters are prepared by the knowngeneral method [Beck J. R., J. Org. Chem., 37:3224 (1972)]. The3-hydroxy-indole-2-carboxylate esters are prepared by known methods suchas Unangst P. C., et al., J.. Heterocyclic Chem., 24:811 (1987) andMoyer M. P., et al., J. Org. Chem., 51:5106 (1986). The 3-thio-indole-2-carboxylate esters are prepared by known methods such as Unangst P.C., et al., J. Heterocyclic Chem., 24:811 (1987); Atkinson J. G., etal., Synthesis, 480 (1988); and Nagarajan K., et al. Indian J. Chem.,20B:672 (1981). The 3-amino-indole-2-carboxylate esters are prepared byknown methods such as Simakov S.V., et al., Khim.-Farm. Zu., 17:1183(1983).

The conversion of compounds of type 1 to those of this invention isshown in Scheme 1. The esters are treated with an α-halo-substitutedacetonitrile derivative such as bromoacetonitrile in the presence of abase such as potassium t-butoxide in tetrahydrofuran, acetonitrile, ordimethylsulfoxide at 0°-80° C. to provide esters of type 2. The nitrilegroup is reduced to the corresponding primary amine and the resultantintermediate 3 is cyclized to the lactam 4. The preferred conversion ishydrogenation of 2 with Raney cobalt catalyst in a solvent such astetrahydrofuran in the presence of a base such as triethylamine atelevated temperature and pressure. Under these conditions 4 is obtaineddirectly from 2. If intermediate 3 is isolated it is cyclized to 4 underbasic, preferably NaOMe in methanol, or acidic conditions, preferablypolyphosphoric acid, at elevated temperatures.

During the synthesis of some of the invention compounds, it may benecessary or desirable to convert reactive groups such as hydroxy,amino, and carboxy, to derivatives which will protect them from unwantedside reactions when a desired reaction is taking place somewhere else inthe molecule. Such protected hydroxy, amino, and carboxy groups arereadily deprotected by conventional methods. Commonly used chemicalmoieties which serve to protect reactive groups such as hydroxy, amino,and carboxy, and methods for their attachment and subsequent removal,are described by Greene and Wuts in Protective Groups in OrganicSynthesis, John Wiley & Sons, Inc., New York, 1991.

For example, a 3-amino, 3-hydroxy, or 3-thio indole, benzothiophene, orbenzofuran (Compound 1 in Scheme 1) can be reacted with aβ-halo-ethyleneamine where the amino group is protected with a suitableprotecting group (PG) such as a t-butoxycarbonyl (Boc) orbenzyloxycarbonyl (Cbz). Reaction under the same conditions as describedabove provides compounds of type 5. Deprotection (i.e., removal of thePG) of 5 under standard conditions, i.e., trifluoroacetic acid oraqueous acid for the removal of the BOC or hydrogenolysis for removal ofthe Cbz, provides compounds of type 3 that are cyclized as notedearlier. Another approach is the reaction of compounds of type 1 withethyleneimine in an alcoholic solvent to directly provide 3 (see:Nagarajan K., et al. Indian J. Chem,, 20B:672 (1981)).

A second general approach (Scheme 2) to compounds of type 4 is from thecorresponding 3-halo derivatives 6. Reaction of 6 with ethylenediamineand cupric oxide in a solvent such as pyridine in the presence of a basesuch as potassium carbonate provides compounds of type 3 where Y is NH(see: Hiremath S. P., et al., Proc. Nat. Acad. Sci., India, 60:367(1990)). Reaction of 6 with cysteamine in a solvent such asdimethylformamide in the presence of a base such as DBU providescompounds of type 3 where Y is S. Reaction of 6 with nitroethanol in asolvent such as tetrahydrofuran in the presence of a base such aspotassium t-butoxide or potassium hydride provides compounds of type 7.Subsequent reduction of the nitro group to an amine leads to compoundsof type 3 where Y is O. In some of the above cases 3 may not be isolatedbut 4 may be obtained directly.

A third general approach (Scheme 3) also utilizes the 3-halo derivatives6. The 3-halo derivative is treated with a primary amine that contains asuitably protected amino, hydroxy or thiol group at the β-position, toform an amide group, providing an intermediate of type 7. Deprotectionfollowed by cyclization leads to compounds of type 4. A similar sequencestarts with the 3-hydroxy, thiol or amino compound adding an amine witha suitable leaving group at the β-position. The resultant intermediatesof type 8 are then cyclized to give 4.

Those compounds of type 4 where X is S and Y is O or NR can be convertedto the corresponding sulfoxide and/or sulfone, 9, with an oxidizingagent such as mochloroperbenzoic acid (m-CPBA) or an oxaziridine withthe reaction conditions determining the extent of oxidation (Scheme 4).For those compounds of type 4 wherein Y is S, similar oxidation wouldprovide either the sulfoxide or sulfone of type 10.

Conditions within the description of Schemes 1 through 4 and variationsin the description are known or can readily be determined from analogousreactions known to one skilled in the art. ##STR3##

The following examples are illustrative of the preparation of thecompounds of the present invention.

EXAMPLE 12,3-Dihydro-9-methoxy[1]benzothieno[2,3-f]-1,4-thiazepin-5(4H)-one

To a room temperature solution of methyl3-chloro-5-methoxy-benzo[b]thiophene-2-carboxylate (500 mg, 1.95 mmols)[prepared by reaction of the known3-chloro-5-methoxy-benzo[b]thiophene-2-carbonyl chloride withmethanol-[J. Med, Chem., 35:958 (1992)]] in 20 mL of DMF is addedcysteamine-HCl (885 mg, 7.79 mmol) followed by DBU (2.33 mL, 15.58mmol). The reaction mixture is stirred at room temperature for 1.5 hoursthen warmed to 70° C. The mixture is diluted with ethyl acetate andwashed with aqueous HCl, water and brine. The organic layer is driedover MgSO₄, filtered and concentrated in vacuo. The crude product isrecrystallized from hexane and ethyl acetate to provide2,3-dihydro-9-methoxy[1]benzothieno[2,3-f]-1,4-thiazepin-5(4H)-one in74% yield; mp 209°-209.5° C.

EXAMPLE 2 2,3-Dihydro-[1]benzothieno[2,3-f]-1,4-oxazepin-5(4 H)-one

A mixture of the methyl ester of3-(cyanomethoxy)benzo[b]thiophene-2-carboxylic acid (405 mg, 1.64 mmols)[J. Hetero. Chem., 12:1037 (1975)], 0.5 mL of Et₃ N and 0.50 g of RaCoin 50 mL of THF is heated at 100° C. under 1200 psi of hydrogen. Thereaction mixture is concentrated in vacuo. Column chromatography elutingwith a gradient of 1:1 hexane:ethyl acetate to all ethyl acetateprovides 2,3-dihydro -[1]benzothieno[2,3-f]1,4-oxazepin-5(4H)-one in 55%yield; mp 244°-245° C.

EXAMPLE 32,3-Dihydro-9-methoxy-[1]benzothieno[2,3-f]-1,4-thiazepin-5(4H)-one-1-oxide

A mixture of2,3-dihydro-9-methoxy-[1]benzothieno[2,3-f]-1,4-thiazepin-5(4H)-one (200mg, 0.75 mmol) and NaBO₃ -4H₂ O(116 mg, 0.75 mmol) in 18 mL of AcOH isstirred at room temperature overnight. The reaction mixture is filteredand 60 mL of water is added to the filtrate. Filtration provides2,3-dihydro-9-methoxy-[1]benzothieno[2,3-f]-1,4-thiazepin-5(4H)-one-1-oxidein 69% yield; mp 215°-216° C. (dec).

EXAMPLE 43,4-Dihydro-9-methoxy-6-methyl-2H-1,4-oxazepino[6,7-b]-indol-5(6H)-one

A. Methyl 3-(cyanomethoxy)-5-methoxy-1-methyl-1H-indole-2-carboxylate

A suspension of potassium tert-butoxide (3.2 g, 29 mmol) in 60 mL ofdimethyl sulfoxide is treated in portions with methyl3-hydroxy-5-methoxy-1-methyl 1H-indole-2-carboxylate (5.6 g, 24 mmol;Unangst P. C., et al., J. Heterocyclic Chem., 24:811 (1987)) . Themixture is stirred for 15 minutes, and chloroacetonitrile (4.8 mL, 5.7g, 76 mmol) is added dropwise. The mixture is heated at 80° for 90minutes, cooled, and added to 800 g of ice and water. The precipitatedsolid is filtered, washed with 10% methanol-water, and recrystallizedfrom aqueous acetonitrile to give 3.9 g (60%) of product; mp 136°-137°C.

B. A mixture of methyl3-(cyanomethoxy)-5-methoxy-1-methyl-1H-indole-2-carboxylate (0.60 g, 2.2mmol) and triethylamine (0.40 mL, 0.29 g, 2.9 mmol) in 35 mL oftetrahydrofuran in a pressure reaction vessel is treated with Raneycobalt catalyst (0.40 g). The reactor is pressurized with hydrogen (590psi) and heated at 80° C. for 10 hours. The cooled reaction mixture isfiltered and the filtrate evaporated. The oil residue is dissolved in 50mL of methanol, and sodium methoxide (0.80 g, 15 mmol) is added to thesolution. The mixture is heated at reflux for 3 hours, then cooled andevaporated. The residue is distributed between 75 mL of ethyl acetateand 150 mL of brine. The aqueous layer is extracted several times withfresh ethyl acetate. The combined organic layers are washed with brine,dried (anhydrous sodium sulfate) and evaporated. The crude productresidue is purified by flash chromatography (silica gel, 5% methanol indichloromethane elution) to yield 0.18 g (33%) of product. A samplerecrystallized from ethyl acetate-hexane has mp 184°-186° C.

EXAMPLE 5 2,3-Dihydro-1H-benzothieno-[3,2-e]-1,4-diazepine-5-one

3-(2-Aminoethylamino)benzo[b]thiophene-2-carboxylic acid methyl esterhydrochloride

A solution of 2-(4,5-dihydro-1H-imidazol-2-yl)benzenethiol (1.00 g, 5.62mmol) [Hegen, H., Fleig, H. Justus Liebigs Ann. Chem. 11:1994 (1975)]and chloromethyl acetate (610 mg, 5.62 mmol) in 15 mL of methanol isheated at reflux for 90 minutes. The reaction is cooled to roomtemperature and filtered. The filtrate is concentrated to dryness andthe residue dissolved in hot chloroform. After several hours theresulting precipitate is collected and dried. The mother liquor affordsa second crop of crystals giving3-(2-aminoethylamino)benzo[b]thiophene-2-carboxylic acid methyl esterhydrochloride in an overall yield of 61%, mp 219°-220° C.

2,3-Dihydro-1H-benzothieno-[3,2-e]-1,4-diazepin-5-one

A solution of 3-(2-aminoethylamino)benzo[b]-thiophene-2-carboxylic acidmethyl ester hydrochloride (339 mg, 1.18 mmol) and freshly preparedsodium methoxide (from 134 mg, 2.48 mmol of sodium) in 5 mL of methanolis heated at reflux for 18 hours. Upon cooling, the reaction isneutralized with 25 mL of 1N HCl and cooled to 0° C. for 1 hour. Theresulting yellow crystalline material is filtered and dried under vacuumat 60° C. for several hours to provide2,3-dihydro-1H-benzothieno-[3,2-e]-1,4-diazepin-5-one in 64% yield.Chromatography, eluting with a gradient of 2% methanol in ethyl acetatein 5% methanol in ethyl acetate, gives analytically pure,2,3-dihydro-1H-benzothieno-[3,2-e]-1,4-diazepin-5-one, mp 210°-212° C.

EXAMPLE 62,3-Dihydro-9-methoxy-1H-benzothieno-[2,3-f]-1,4-oxazepin-5-one

3-Cyanomethoxy-5-methoxy-benzo[b]thiophene-2-carboxylic acid methylester

To a room temperature solution of methyl3-hydroxy-5-methoxybenzo[b]thiophene-2-carboxylate (1.00 g, 4.2 mmol)[Connor, et al., J. Med. Chem. 35:959 (1992)]in 20 mL of DMSO is addedpotassium t-butoxide (494 mg, 4.41 mmol) followed by bromoacetonitrile(878 μL, 12.58 mmol). The mixture is stirred at room temperature for 1.5hours, then poured into ethyl acetate and 1N HCl. The organic layer iswashed with 1N HCl, followed by several portions of brine, and driedover MgSO₄. Filtration followed by removal of solvent in vacuo andrecrystallization of the residue from ethyl acetate:hexane gives 413 mg.additional crop of 112 mg can be obtained from the mother liquor, mp159.5°-160° C.

2,3-Dihydro-9-methoxy-1H-benzothieno-[2,3-f]-1,4-oxazepin-5-one

A solution of 3-cyanomethoxy-5-methoxy-benzo[b]-thiophene-2-carboxylicacid methyl ester (2.5 g, 9.0 mmol) in 50 mL of THF is heated tovigorous reflux. Borane-dimethyl sulfide (9.0 mL, 90.2 mmol) is rapidlyadded and heating continued for 25 minutes with THF being added as itevaporates. An additional amount of borane-dimethyl sulfide (4.0 mL) isadded and heating continued for 10 minutes. The reaction mixture iscooled to 0° C. and 50 mL of 6N HCl is carefully added. Hydrogen gas isevolved and the temperature of the reaction mixture increases. Theresultant precipitate is collected by filtration, washed with water, anddried in vacuo overnight.

The solid (2.3 g, 8.2 mmol) is added to a freshly prepared solution ofsodium methoxide (from 1.9 g, 82.0 mmol of sodium) in 40 mL of methanol.The reaction mixture is warmed to 50° C. for 2 hours, then heated atreflux for 2 hours. After cooling to room temperature, the precipitateis collected and washed with cold methanol, followed by cold diethylether. The solid is dried in vacuo overnight to give 1.18 g (52%). Ananalytical sample of2,3-dihydro-9-methoxy-1H-benzothieno-[2,3-f]-1,4-oxazepin-5-one isobtained by recrystallization from ethyl acetate:hexane, mp 264°-265° C.

EXAMPLE 7

2,3-Dihydro-9-methoxy-6-oxide-1H-benzothieno-[2,3-f]-1,4-oxazepin-5-one

To a suspension of2,3-dihydro-9-methoxy-1H-benzothieno-[2,3-f]-1,4-oxazepin-5-one (1.00 g,4.0 mmol) in 100 mL of warm methanol is added 30% hydrogen peroxide (8.0mL, 80 mmol) followed by selenium dioxide (445 mg, 4.01 mmol). Thereaction mixture is stirred at room temperature for 3 hours then heatedat 30° C. for 1.5 hours followed by heating at 40° C. for 2 hours. Thereaction mixture is cooled to -40° C. and the resulting precipitate iscollected by filtration. The residue is chromatographed elutinginitially with 5% methanol in ethyl acetate gradually increasing thesolvent polarity to 1:1 methanol:ethyl acetate to give 338 mg ofproduct. An analytical sample of2,3-dihydro-9-methoxy-6-oxide-1H-benzothieno-[2,3-f]-1,4-oxazepin-5-oneis obtained by recrystallization from methol:ethyl acetate, mp 273°-274°C.

EXAMPLE 8 2,3-Dihydro-9-methoxy-2-methyl-1H-benzothieno-[2,3-f]-1,4-oxazepin-5-one

3-(1-Cyanoethoxy)-5-methoxy-benzo[b]thiophene-2-carboxylic acid methylester

To a room temperature solution of methyl3-hydroxy-5-methoxybenzo[b]thiophene-2-carboxylate (1.00 g, 4.2 mmol)[Connor, et al., J. Med. Chem. 35:958 (1992)] in 20 mL of DMSO is addedpotassium t-butoxide (494 mg, 4.41 mmol) followed by2-chloropropionitrile (1.1 mL, 12.6 mmol). The mixture is stirred atroom temperature for 1.5 hours then warmed to 82° C. for 3 hours. Thereaction mixture is poured into ethyl acetate and 1N HCl. The organiclayer is washed with 1N HCl, followed by several portions of brine anddried over MgSO₄. Filtration followed by removal of solvent in vacuo andrecrystallization of the residue from ethyl acetate:hexane gives 853 mg,mp 127°-129° C.

2,3-Dihydro-9-methoxy-2-methyl-1H-benzothieno-[2,3-f]-1,4-oxazepin-5-one

A solution of 3-(1-cyanoethoxy)-5-methoxybenzo[b]-thiophene-2-carboxylicacid methyl ester (400 mg, 1.37 mmol) in 10 mL of THF is heated tovigorous reflux. Borane-dimethyl sulfide (1.4 mL, 13.7 mmol) is addeddropwise and heating continued for 20 minutes with THF being added as itevaporates. The reaction mixture is cooled to room temperature and 7.5mL of 6N HCl is carefully added. After 5 minutes the reaction mixture iscooled to 0° C. and 68.5 mL of 1N NaOH is added followed by ethylacetate. The layers are separated and the organic phase is washed with1:1 brine:water, then with additional brine. The organic phase is driedover MgSO₄, filtered, and concentrated in vacuo. The residue ischromatographed eluting with a gradient of 5:25:70methanol:hexane:chloroform to 10:90 methanol:chloroform to 30:70methanol:chloroform to give 135 mg of product. An analytical sample of2,3-dihydro-9-methoxy-2-methyl-1H-benzothieno-[2,3-f]-1,4-oxazepin-5-one is obtained by recrystallization from ethylacetate:hexane, mp 185°-186° C.

The invention compounds are readily formulated with common diluents andcarriers for convenient oral or parenteral administration to humans andanimals for treatment of diseases such as inflammation, especiallyarthritis and the like. The following examples illustrate thepreparation of typical pharmaceutical formulations.

EXAMPLE 9 Preparation of 250-mg Capsule

2,3-Dihydro-9-isopropoxy-7-chloro-1H-benzothieno-[2,3-f]-1,4-oxazepin-5-one(250 mg), is blended to uniformity with 150 mg of lactose and 150 mg ofcorn starch. The mixture is encapsulated into gelatin capsules. Suchcapsules are orally administered at the rate of one to three each dayfor treatment of arthritis.

    ______________________________________                                        EXAMPLE 10                                                                    Formulation for Oral Suspension                                               Ingredient               Amount                                               ______________________________________                                        2,3-Dihydro-8-ethyl-10-trifluoro-                                                                      500 mg                                               methyl-6-oxide-1H-benzothieno-                                                [2,3-f]-1,4-oxazepin-5-one                                                    Sorbitol solution (70% N.F.)                                                                            40 mL                                               Sodium benzoate          150 mg                                               Saccharin                 10 mg                                               Cherry Flavor             50 mg                                               Distilled Water q.s. ad. 100 mL                                               ______________________________________                                    

The sorbitol solution is added to 40 mL of distilled water and theoxazepinone is suspended thereon. The saccharin, sodium benzoate, andflavoring are added and dissolved. The volume is adjusted to 100 mL withdistilled water. Each milliliter of syrup contains 5 mg of theoxazepinone. This oral formulation is ideally suited for treatinginflammation in pediatric care.

EXAMPLE 11 Preparation of Parenteral Solutions

In a solution of 700 mL of propylene glycol and 200 mL of distilledwater for injection is dissolved 20.0 g of2,3-dihydro-7-dimethylamino-1H-benzothieno-[3,2-e]-1,4-diazepin-5-one.The pH of the solution is adjusted to 5.5 with hydrochloric acid, andthe volume is made up to 1000 mL with distilled water. The formulationis sterilized, filled into 5.0 mL ampoules each containing 2.0 mL(representing 40 mg of active diazepinone) and sealed under nitrogen.The formulation is administered intravenously to patients suffering frominflammation or AIDS.

EXAMPLE 12 Preparation of Topical Cream

Five hundred milligrams of2,3-dihydro-7-ethoxybenzofurano-[2,3-f]-1,4-oxazepin-5-one is mixed with15 g of cetyl alcohol, 1 g of sodium lauryl sulfate, 40 g of liquidsilicone D.C. 200 (sold by Dow Corning Co., Midland, Mich.), 43 g ofsterile water, 0.25 g of methylparaben, and 0.15 g of propylparaben. Themixture is warmed to about 75° C. with constant stirring, and thencooled to room temperature at which it congeals. The preparation isapplied to the skin surface of a person suffering from inflammation.

We claim:
 1. A method for treating inflammatory disease in humanscomprising administering an antiinflammatory amount of a compound of theformula ##STR4## or a pharmaceutically acceptable acid addition saltthereof, wherein R₁, R₂, R₃, and R₄ are each independently hydrogen,hydroxy, halogen, lower alkyl, lower alkoxy, benzyloxy, trifluoromethyl,nitro, or --NR₈ R₉, in which R₈ and R₉ are each independently hydrogenor lower alkyl;R₅ and R₆ are each independently hydrogen, lower alkyl orphenyl; X is O, S(O)_(n) or NR₇ ; Y is O, S(O)_(n) or NR₈ ; R₇ ishydrogen, lower alkyl, phenyl, benzyl, CH₂ R₉ or lower alkyl, phenyl,benzyl substituted with halo; R₈ is hydrogen, lower alkyl or phenyl; nis an integer of 0, 1 or 2;with the provisos that 1) when X is NH, Y isNH, R₁ is H, R₃ is H and R₄ is Br, R₂ is not methyl; 2) when X is NH, Yis NH, R₁, R₃ and R₄ are H, R₁ is not methoxy or ethoxy, and 3) when Xis NH, Y is S, at least one of R₁, R₂, R₃ and R₄ is not H.
 2. A methodaccording to claim 1 employing a compound wherein R₁, R₃ and R₄ arehydrogen.
 3. A method according to claim 2 employing a compoundwhereinR₂ is hydrogen or lower alkoxy; X is O, S(O)_(n) or NR₇ ; Y is Oor S(O)_(n) ; R₇ is hydrogen or lower alkyl, and n is 0, 1 or
 2. 4. Amethod according to claim 3 employing a compound selectedfrom2,3-dihydro-9-methoxy-[1]benzothieno[2,3-f]-1,4-thiazepin-5(4H)-one;2,3-dihydro-[1]benzothieno-[2,3-f]-1,4-oxazepin-5(4H)-one;2,3-dihydro-9-methoxy-[1]benzothieno[2,3-f]-1,4-thiazepin-5(4H)-one-1-oxide3.4-dihydro-9-methoxy-6-methyl-2H-1,4-oxazepino[6,7-b]-indol-5(6H)-one;2,3-dihydro-1H-benzothieno-[3,2-e]-1,4-diazepine-5-one;2,3-dihydro-9-methoxy-1H-benzothieno-[2,3-f]-1,4-oxazepin-5-one;2,3-dihydro-9-methoxy-6-oxide-1H-benzothieno-[2,3-f]-1,4-oxazepin-5-one;or2,3-dihydro-9-methoxy-2-methyl-1H-benzothieno-[2,3-f]-1,4-oxazepin-5-one.